Lab on a chip devices continuously strive to provide more information and function in ever smaller packages. This becomes evident when considering the growth of on-chip microarrays and micro/nanoreactors for massively parallel or high-throughput processing. On-chip nanowells and droplet reactors have gained attention in the field of digital biology for applications like single-cell analysis and single-copy nucleic acid detection including: digital quantification of DNA, monoclonal template amplification for bead-based gene sequencing, reverse transcriptase (RT-PCR) for detection of single RNA fragments, and multiplexed digital PCR. Digital PCR platforms benefit from each reactor having its own microenvironment where the amplification in one reactor does not interfere with that of another. This provides a digital output of nucleic acid concentrations with increased quantitative resolution.
Furthermore, single DNA molecules can be quantified even in the presence of competing template sequences, which would otherwise skew traditional qPCR results. Unfortunately, the dynamic-range of on-chip digital PCR lags behind its analog counterparts by three to four orders of magnitude due to the lack of compartmentalized reactors and limited volume throughput. This is further compounded by Poisson distribution behavior of single molecule encapsulation, which requires low concentrations of DNA relative to reactor number. In addition, many nucleic acid processing schemes benefit from real-time fluorescence measurements, which provide temporal information about PCR amplification, but few high throughput platforms provide this ability.
A prior method that has been used was to compare the stoichiometric amplification efficiency of PCR and determine the initial concentration based on cycle number when fluorescence exceeded a threshold value. Another approach that has been used was monitoring real-time PCR amplification of nucleic acids to estimate amplification based on intrinsic amplification rates corresponding to the amount of time or number of cycles over which replication occurred. As described below, the device and methods described herein are able to yield significantly higher dynamic ranges with exceptionally higher resolution and accuracy than any prior methods.